蓝藻橙色类胡萝卜素蛋白的基因克隆及在大肠杆菌中的异源表达和功能分析

Gene Cloning of the Orange Carotenoid Protein from Cyanobacteria,and Its Ectopic Expression and Functional Evaluation in Escherichia coli

旨在体内证明蓝藻橙色类胡萝卜素蛋白（OCP）具备不依赖类胡萝卜素的单线态氧（~1O_2）清除功能。通过PCR扩增蓝藻OCP基因,对其进行原核诱导表达,并通过细菌点板和生长曲线测定法检验它在大肠杆菌中的抗~1O_2活性。结果表明,直接从蓝藻水体提取的宏基因组中成功扩增得到OCP基因SyOCP,其编码区大小为954 bp。生物信息学分析表明,SyOCP来源于主要蓝藻种类——集胞藻（Synechocystis sp.PCC 6803）。经IPTG诱导,SyOCP基因在大肠杆菌中获得了高水平的可溶性表达。另外,异源表达SyOCP的重组大肠杆菌对常见的~1O_2光敏诱发剂染料——亚甲基蓝的耐受性得到了显著增强。蓝藻OCP（不结合类胡萝卜素）内在的抗~1O_2功能在大肠杆菌中得到了体内验证。

The aim is to verify the orange carotenoid protein（OCP）in cyanobacteria can scavenge singlet oxygen（~1O_2）independentof carotenoids in vivo.The cyanobacterial OCP gene was amplified by PCR,and then subjected to prokaryotic inducible expression and furtherevaluation of anti-~1O_2 activity in Escherichia coli by bacterial dot-plating test and growth curve assay.Results showed that the gene（Sy OCP）of OCP was successfully amplified from the metagenome of the cyanobacteria water,with a coding region of 954 bp.Bioinformatics analysisindicated that Sy OCP was from the major cyanobacteria species—Synechocystis sp.PCC 6803.By IPTG induction,the Sy OCP gene washighly expressed in E.coli with soluble protein.In addition,the ectopic expression of Sy OCP in recombinant E.coli resulted in the notableenhancement of it on the tolerance to the common ~1O_2-induced photosensitizer dye,methylene blue.The intrinsic anti-~1O_2 role of cyanobacterialOCP（without binding carotenoids）was in vivo verified in E.coli.